Les travaux ont été soutenus par des subventions non. 01GU0615 du «Bundesministerium für Forschung und Technologie (BMBF).

1) Livraison bactérienne des shRNA <ol><li> Avant de commencer avec la culture bactérienne, il faut se préparer milieu LB et LB-agar. </li><li> Pour le milieu LB peser extrait de levure (0,5% p / v), Bacto tryptone (1,0% p / v), et du NaCl (0,6% p / v) et diluer ces composants dans de l&#39;eau bidest. Les solutions préparées doivent être stérilisés dans un autoclave et sont alors prêtes à utiliser. </li><li> Pour LB-agar gélose Bacto (1,5% p / v) doit être ajoutée à la précédente milieu LB à la stérilisation. </li><li> Chauffer LB-agar dans le micro-ondes jusqu&#39;à ce que le LB-agar soit complètement dissoute. Laisser la solution refroidir jusqu&#39;à ce que la bouteille peut être touché facilement sans se brûler. Ajouter à la kanamycine (100 mg / ml) pour la sélection de clones positifs dans d&#39;autres mesures. </li><li> Préparer LB-agar sur un banc à flux laminaire (conditions stériles) par pipetage 20 ml de la solution de LB-agar en 10 cm de Petri-plats. Laissez les plaques refroidir jusqu&#39;à ce le liquide est devenu solide. Les plaques sont maintenant prêts à utiliser et peut être conservé à 4 ° C. </li><li> Des vecteurs d&#39;expression ShRNA-encodage sont transformées en compétents E. coli ceq221 par choc thermique utilisant la procédure de CaCl 2. </li><li> Plaque les bactéries transformées sur LB-agar contenant 100 ug / ml de kanamycine, fermez le couvercle, sceller les plaques avec du parafilm et cultiver l&#39;envers à 37 ° C pendant la nuit. </li><li> Inoculer les cultures bactériennes mini clones positifs en choisissant colonies cultivées avec un cure-dent. Transférer le cure-dent dans 7,0 ml de milieu LB frais contenant 100 mg / ml de kanamycine et de cultiver à 37 ° C pendant la nuit sur un agitateur à 200 tpm. </li><li> Inoculer 100 ml de milieu LB frais contenant 100 mg / ml de kanamycine 1:100 avec la culture nuit sur un mini dans un flacon de 1 litre Erlenmeyer, et incuber à 37 ° C pendant la nuit sur un agitateur à 200 tpm. </li><li> Graine de 25 x 10 4 (nombre de cellules ensemencées dépend de la vitesse de croissance des cellules de la lignée cellulaire selon; confluence devrait être ~ 70-80% 24 heures après l&#39;ensemencement) des cellules de carcinome gastrique (EPG85-257RDB) par puits dans 6 - plats puits et incuber ces à 37 ° C, dans une atmosphère de 5% de CO 2, vapeur d&#39;eau saturée pendant la nuit. </li><li> Précédente à l&#39;infection des cellules cancéreuses, sur les cultures nuit du tk ARNi contenant le vecteur E. coli sont mesurés dans un photomètre pour déterminer la DO 600. Diluer la solution bactérienne jusqu&#39;à une densité optique de 0,5 est atteint (DO600 = 0,5 équivaut à 1,6 x 10 8 cellules bactériennes / ml). </li><li> Remplacez le support SVF contenant des cellules de culture des cellules carcinomes contre la FCS milieu sans précédent pour 30 min bactérienne co-incubation. </li><li> Laver deux fois avec des bactéries 1 x PBS, et diluer dans le sérum sans Leibovitz milieu L-15. </li><li> Ajouter des bactéries aux cellules cancéreuses diluée au désirée MOI (multiplicité d&#39;infection = nombre de cellules bactériennes par des cellules cancéreuses) et co-incubation des bactéries et des cellules cancéreuses pendant 2 heures à 37 ° C. </li><li> Après 2 heures de cellules de cancer co-incubation ont été lavés deux fois avec du PBS 1x x et une fois avec du sérum contenant Leibovitz L-15 milieu supplémenté avec 100 U / ml de pénicilline, 100 pg / ml de streptomycine, 2,5 pg / ml d&#39;amphotéricine, 150 ug / ml de gentamicine, et 100 pg / ml de kanamycine. </li><li> Continuer à cultiver les cellules cancéreuses à 37 ° C, dans une atmosphère de 5% de CO 2, la vapeur d&#39;eau saturée. </li><li> Les effets du traitement bactérienne peut être visualisé par microscopie à fluorescence, mesurée par PCR quantitative en temps réel sur le niveau d&#39;ARNm RT, et par analyse western blot sur le niveau de protéines, et indiqué avec des dosages fonctionnels tels que l&#39;analyse FACS, et les tests de prolifération cellulaire. (Si voulu, ces procédures peuvent être décrites en détail). </li></ol> Résultats Représentant 2) Si le protocole est réalisé correctement, les résultats devraient être comparables à celles indiquées ci-dessous. La microscopie à fluorescence La figure 1 montre un carcinome gastrique humaine trois heures après le traitement bactérienne (a) en comparaison à une cellule non traitée (b). Autour du noyau de la cellule traitée, les bactéries peuvent être détectées. <img src="/files/ftp_upload/2099/2099fig1.jpg" alt="Figure 1" /> Figure 1: Fluorescent microscope de cellules humaines carcinome gastrique après traitement bactérien (1:500) et d&#39;une cellule humaine non traitée carcinome gastrique comme témoin, DAPI-coloration, passe-bande DAPI filtre (Λem = 640 nm), objectif 40x. Quantitative en temps réel RT-PCR Dans la figure 2 une régulation négative de l&#39;ARNm du gène MDR1 d&#39;environ 70% (faisceau noir) peuvent être observés après traitement avec la bactérie thérapeutiques. Cellules parentales servir de contrôle positif en raison de l&#39;absence d&#39;une surexpression du gène MDR1. La lignée cellulaire 257RDB p170 contenant un plasmide exprimant anti-MDR1 shRNA s pris comme comparaison directe de la technologie ARNi pour transkingdom R d&#39;autresNAi taire stratégies. La lignée cellulaire résistante traitée EPG85-257RDB surexprimant MDR1, la même lignée cellulaire traités avec des bactéries qui n&#39;ont pas les shRNA plasmide exprimant, et cette lignée de cellules traitées avec des bactéries thérapeutiques portant un plasmide exprimant anti-MRP2 shRNA ont été pris comme contrôles positifs. <img src="/files/ftp_upload/2099/2099fig2.jpg" alt="Figure 2" /> Figure 2: quantitative en temps réel RT-PCR l&#39;expression des ARNm du gène MDR1 après le traitement avec des produits thérapeutiques E. coli exprimant ceq221 anti-MDR1 shRNA (MOI 1: 500). Normalisation a été réalisée en utilisant l&#39;aldolase gène de ménage. Le ratio MDR1/aldolase des cellules non traitées de la lignée cellulaire EPG85-257RDB ont été de 100%. P-valeurs ont été calculées à l&#39;aide de l&#39;étudiant t-test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Western blot La figure 3 montre que le MDR1 la régulation négative a également eu lieu sur le niveau de protéine bactérienne après le traitement. Elle montre le manque d&#39;expression du gène MDR1 de la drogue lignée cellulaire parentale sensibles (EPG85-257P), l&#39;expression du gène MDR1 de la lignée cellulaire de drogue traitées résistant (EPG85-257RDB) et l&#39;expression du gène MDR1 de l&#39;échantillon traité. Une réglementation claire en bas de MDR1 des cellules des bactéries traitées peuvent être observés. <img src="/files/ftp_upload/2099/2099fig3.jpg" alt="Figure 3" /> Figure 3: Analyse par Western blot MDR1 niveaux d&#39;expression des traités sensible aux médicaments EPG85-257P, la non traité résistant aux médicaments EPG-257RDB et des EPG85-257RDB après co-incubation avec E.. coli ceq221 + p43 MDR1. Ab primaire C219 1:100, et anti-actine 1:5 000, secondaire Ab anti-souris 1:10 000. Test de cytotoxicité L&#39;analyse fonctionnelle comme le test de cytotoxicité montre la figure 4 sont également des indicateurs pour le fonctionnement de l&#39;ARNi transkingdom. La lignée cellulaire parentale et la lignée cellulaire contenant un shRNA anit-MDR1 plasmide exprimant ne montrent aucune résistance à la daunorubicine. La lignée cellulaire résistante EPG85-257RDB et deux contrôles supplémentaires ne montrent pas des changements importants dans la résistance en comparaison de l&#39;échantillon traité avec des bactéries shRNA anti-MDR1 exprimer où la résistance à la daunorubicine pu être renversé par environ 90%. <img src="/files/ftp_upload/2099/2099fig4.jpg" alt="Figure 4" /> Figure 4: test de cytotoxicité. Médicaments spécifiques IC50-valeurs déterminées par un essai de cytotoxicité à la survie cellulaire. P-valeurs ont été calculées à l&#39;aide de l&#39;étudiant t-test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001). Dosage de l&#39;accumulation d&#39;anthracyclines Selon la résistance des bactéries abaissé d&#39;échantillons traités (figure 4), l&#39;accumulation de cellules anthracycline shRNA anti-MDR1 traitée est augmenté d&#39;environ 90% comme indiqué dans la figure 5. La lignée cellulaire parentale et la lignée cellulaire 257RDB p170 montrent une forte accumulation daunorubicine jusqu&#39;à 100%. La variante résistante montre rarement toute accumulation. Les cellules traitées avec des bactéries shRNA anti-MDR1 exprimer montrent une augmentation de l&#39;accumulation anthracycline de 90%. <img src="/files/ftp_upload/2099/2099fig5.jpg" alt="Figure 5" /> Figure 5: dosage de l&#39;accumulation des anthracyclines. L&#39;accumulation des anthracyclines de cellules de carcinome 6 jours après le traitement bactérienne mesurée par cytométrie en flux. P-valeurs ont été calculées à l&#39;aide de l&#39;étudiant t-test (* = p &lt;0,05, ** = p &lt;0,005, *** = p &lt;0,001).

<ol>
	<li>Kr&#252;hn, A., Wang, A., Fruehauf, J. H., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs+by+transkingdom+RNA+interference+modulates+the+classical+ABCB1-mediated+multidrug-resistant+phenotype+of+cancer+cells.">Delivery of short hairpin RNAs by transkingdom RNA interference modulates the classical ABCB1-mediated multidrug-resistant phenotype of cancer cells.</a> Cell Cycle. 8, 3349-3354 (2009).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDR1/P-glycoprotein+(ABCB1)+as+target+for+RNA+interference-mediated+reversal+of+multidrug+resistance.">MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated reversal of multidrug resistance.</a> Curr. Drug Targets. 7, 813-821 (2006).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+cancer+multidrug+resistance:+a+still+unsolved+problem.">An overview of cancer multidrug resistance: a still unsolved problem.</a> Cell. Mol. Life Sci. 65, 3145-3167 (2008).</li><li>Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA+interference+in+drug-resistant+cancers.">Therapeutic potential of RNA interference in drug-resistant cancers.</a> Fut. Oncol. 5, 169-185 (2009).</li><li>Nguyen, T. A., Fruehauf, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transkingdom+RNA+interference+(tkRNAi):+a+novel+method+to+induce+therapeutic+gene+silencing.">Transkingdom RNA interference (tkRNAi): a novel method to induce therapeutic gene silencing.</a> Methods Mol. Biol. 514, 27-34 (2009).</li><li>Nieth, C., Priebsch, A., Stege, A., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+classical+multidrug+resistance+(MDR)+phenotype+by+RNA+interference+(RNAi.">Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi.</a> FEBS Lett. 545, 144-150 (2003).</li><li>Stege, A., Priebsch, A., Nieth, C., Lage, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+and+complete+overcoming+of+MDR1/P-glycoprotein-mediated+multidrug+resistance+in+human+gastric+carcinoma+cells+by+RNA+interference.">Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference.</a> Cancer Gene Ther. 11, 699-706 (2004).</li><li>Stein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+in+vivo+reversal+of+the+multidrug+resistance+(MDR)+phenotype+by+jet-injection+of+anti-MDR1+short+hairpin+RNA-encoding+plasmid+DNA.">Complete in vivo reversal of the multidrug resistance (MDR) phenotype by jet-injection of anti-MDR1 short hairpin RNA-encoding plasmid DNA.</a> Mol. Ther. 16, 178-186 (2008).</li><li>Xiang, S., Fruehauf, J., Li, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-expressing+bacteria+elicit+RNA+interference+in+mammals.">Short hairpin RNA-expressing bacteria elicit RNA interference in mammals.</a> Nat. Biotechnol. 24, 697-702 (2006).</li></ol>

Le nombre de cellules ensemencées pour l&#39;infection et la MOI correspondants utilisés, dépendent essentiellement sur les lignées cellulaires sous observation et leur vitesse de croissance. Pour trouver le nombre de cellules optimales pour le semis, pré-expériences pour déterminer la vitesse de croissance sont fortement recommandées. Outre cela, IAM de différents doivent être testés en raison de la limite d&#39;étendre à laquelle les cellules peuvent se l&#39;invasion bactérienne sans mourir de stress. L...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>214050</td>
<td>&nbsp;</td>
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2612</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Phosphate Buffered Saline-PBS (10 x)</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>14080048</td>
<td>&nbsp;</td>
<tr>
<td>E. coli ceq221</td>
<td>&nbsp;</td>
<td>Cequent Pharmaceuticals Inc.</td>
<td>&nbsp;</td>
<td>No catalogue available, direct order</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>&nbsp;</td>
<td>Biochrom AG</td>
<td>A2710</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen GmbH</td>
<td>&nbsp;</td>
<td>Contains 5,000 units of penicillin (base) and 5,000 &mu;g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline.</td>
</tr>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Merck KgaA</td>
<td>1064060500</td>
<td>&nbsp;</td>
<tr>
<td>Tryptone</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>211705</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Difco Laboratories</td>
<td>212750</td>
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bacterial delivery of rnai effectors: transkingdom rnai

L&#39;interférence ARN (ARNi) représente un mécanisme de haute efficace pour l&#39;inhibition spécifique de l&#39;expression de l&#39;ARNm. Outre son potentiel comme outil de laboratoire puissant, la voie de l&#39;ARNi semble être prometteuse pour l&#39;utilisation thérapeutique. Pour le développement de l&#39;interférence ARN (ARNi) basé sur les thérapies, la livraison de l&#39;ARNi médiation des agents à des cellules cibles est l&#39;un des obstacles majeurs. Une nouvelle stratégie pour surmonter cet obstacle est transkingdom ARNi (tk ARNi). Cette technologie utilise des bactéries non pathogènes, par exemple, Escherichia coli, pour produire et livrer en épingle à cheveux courts ARN thérapeutiques (shRNA) dans les cellules cibles pour induire l&#39;ARNi. Une première génération tk ARNi médiation vecteur, TRIP, contient le promoteur du bactériophage T7 pour régulation de l&#39;expression d&#39;un shRNA thérapeutiques d&#39;intérêt. Par ailleurs, TRIP a le locus Inv partir de Yersinia pseudotuberculosis qui encode invasine, qui permet naturelle des bactéries non invasives d&#39;entrer β1-intégrine-positif cellules de mammifères et le gène HlyA partir de la bactérie Listeria monocytogenes, qui produit listériolysine O. Cet enzyme permet le shRNA thérapeutiques pour échapper à d&#39;entrée des vésicules dans le cytoplasme de la cellule cible. Construit TRIP sont introduits dans une compétents souche non pathogène Escherichia coli, qui code T7 RNA polymérase nécessaire à l&#39;promoteur T7 axée synthèse de shRNA. Une molécule cible bien caractérisé associée au cancer pour les stratégies de RNAi différents ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). Ce ABC-transporteur agit comme une pompe d&#39;extrusion de drogue et médiatise la &quot;classique&quot; multirésistance ABCB1 médiation (MDR) phénotype des cellules cancéreuses humaines qui se caractérise par un profil de résistance croisée spécifique. ABCB1 exprimant différentes cellules cancéreuses ont été MDR traités par anti-ABCB1 shRNA vecteur d&#39;expression portant E. coli. Cette procédure a abouti à l&#39;activation des voies de l&#39;ARNi dans les cellules cancéreuses et une réglementation considérable bas de l&#39;ARNm de ABCB1 encodage ainsi que la pompe d&#39;extrusion médicament correspondant. En conséquence, l&#39;accumulation du médicament a été renforcée dans les cellules du cancer primitif résistantes aux médicaments et le phénotype MDR a été inversé. Au moyen de ce modèle les données fournissent la preuve de concept que les savoirs traditionnels ARNi est adapté à la modulation de cancer associés à des facteurs, par exemple ABCB1 dans des cellules cancéreuses humaines.

Watch this Scientific Journal Video about Bacterial Delivery of RNAi Effectors: Transkingdom RNAi at JoVE.com

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Pour le développement de l&#39;interférence ARN (ARNi) thérapies basées sur une nouvelle stratégie a été développée, transkingdom ARNi (tkRNAi). Cette technologie utilise des bactéries non pathogènes pour produire et livrer en épingle à cheveux courts ARN thérapeutiques (shRNA) dans les cellules cibles. Ici, tkRNAi a été appliquée avec succès pour le renversement de la multirésistance classique ABCB1 médiation (MDR) des cellules cancéreuses.

Livraison bactérienne de l&#39;ARNi Effectors: Transkingdom ARNi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

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Biology

13.4K vues.  Charité Campus Mitte. Pour le développement de l'interférence ARN (ARNi) thérapies basées sur une nouvelle stratégie a été développée, transkingdom ARNi (tkRNAi). Cette technologie utilise des bactéries non pathogènes pour produire et livrer en épingle à cheveux courts ARN thérapeutiques (shRNA) dans les cellules cibles. Ici, tkRNAi a été appliquée avec succès pour le renversement de la multirésistance classique ABCB1 médiation (MDR) des cellules cancéreuses.

Vidéo: Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

Protocol for RNAi Assays in Adult Mosquitoes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite.  The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocol for RNAi Assays in Adult Mosquitoes A. gambiae

Reverse genetic approaches have proven extremely useful for determining which genes underly resistance to vector pathogens in mosquitoes. This video protocol illustrates a method used by the Dimopoulos lab to inject dsRNA into Anopheles gambiae mosquitoes, which harbor the malaria parasite. The technique manipulating the injection setup and injecting dsRNA into the thorax is illustrated.

Protocole pour des essais de l&#39;ARNi dans les moustiques adultes (A. gambiae)

Reverse genetic approaches have proven extremely useful for determining  which genes underly resistance to vector pathogens in mosquitoes.  This video ...

Recherche

Biologie

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 &#181;m and extends over ~15 &#181;m around the mean penetration depth, which is adjustable between 0 and 50 &#181;m. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.

In this video, we show a procedure for an accurate biolistic delivery of reagents into live tissue with a novel miniature gene gun. We are knocking down the expression of the axon guidance molecule Netrin in leech embryos by delivering molecules of dsRNA into the ventral body wall and ganglia of single segments.

Localisé ARNi et l&#39;expression des gènes extra-utérine dans la sangsue médicinale

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identification des phénotypes inhibition de la croissance induite par l&#39;expression de bactéries de type III Effectors dans la levure

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

EsiRNA MISSION pour le dépistage de l&#39;ARNi dans les cellules de mammifères

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

Interférences ARNi par injection dans les ARNdb Drosophile Embryons

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

ARNi Mediated inactivation génique et la transgénèse par microinjection dans le nématode Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

Dépistage ARNi pour identifier les phénotypes postembryonnaires dans C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

Quantitatifs et automatisé à haut débit du génome à l&#39;échelle écrans ARNi dans C. elegans</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. 
This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis1. 
The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

RNAi-mediated Gene Knockdown and In Vivo Diuresis Assay in Adult Female Aedes aegypti Mosquitoes

In this protocol we combine RNAi-mediated gene silencing with an in vivo diuresis assay to study the effects knockdown of genes of interest has on mosquito fluid excretion.

Inactivation génique médiée par ARNi et<em&gt; In Vivo</emEssai diurèse&gt; Femmes adultes<em&gt; Aedes aegypti</em&gt; Les moustiques

This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion ...

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Phosphopeptide Analyse des rongeurs spermatozoïdes épididymaires

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...